Methods

We conducted immunohistochemical analyses of protein expression in primary tumors of patients with early breast cancer and analyzed their association with standard prognostic parameters and clinical outcomes, including local relapse, metastatic recurrence, disease-free survival (DFS), metastasis-free survival (MFS), and overall survival (OS).

Results

Elevated levels of TRAIL-R3 and chemokine (C-C motif) receptor 2 (CCR-2) in TEpCs and OPG and CCL-2 in stromal cells were significantly associated with a higher risk of metastasis (p = 0.032, p = 0.003, p = 0.038, and p = 0.049; respectively). Moreover, high expression of TRAIL-R3 and CCR-2 in TEpCs was associated with shorter DFS, MFS, and OS. High TRAIL-R3 expression in TEpCs was an independent prognostic factor for DFS and OS, and high CCR-2 expression in these cells was an independent prognostic factor for MFS.

Conclusions

High levels of TRAIL-R3 and CCR-2 expression in TEpCs identified patients with early breast cancer with poor outcomes.

Keywords

Background

Breast cancer is the most common cancer among women worldwide [1–4] and in Argentina affects more than 25,000 women and causes more than 5000 deaths each year (Bureau of Health Information Statistics and Nation, Department of Statistics and Health Information, Ministry of Health, Argentina, 2013). Distant metastasis is the main cause of death in these patients [5]. In high-income countries, breast cancer is usually diagnosed early, and treatment with curative intent and manageable toxicity is feasible. However, many women experience recurrence despite receiving optimal therapy, likely because the tumor microenvironment plays a key role in the development of resistance to treatment [6].

Breast cancer tissue comprises tumor epithelial cells (TEpCs) and stromal cells such as mesenchymal stem cells, tumor-associated fibroblasts, fibroblasts, endothelial cells, adipocytes, and immune cells. The interaction of malignant and non-malignant cells influences tumorigenesis, tumor growth, metastasis, and response to therapy [6–15]. Our group demonstrated that spindle-shaped stromal cells are not associated with the vasculature and TEpCs from primary invasive ductal breast cancer in women with stage I or II express molecules such as osteoprotegerin (OPG), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), receptor activator of nuclear factor kappa B ligand (RANKL), stromal cell derived factor (SDF)-1, interleukin (IL)-6, chemokine (C-C motif) ligand-2 (CCL-2) and their receptors [15]. These molecules, which are likely involved in the interactions between these cell types, mediate proliferation, survival, migration, and intravasation of TEpCs as well as angiogenesis in the primary tumor [15]. These findings led us to ask whether the levels of expression of these ligand-receptor pairs are useful for predicting the outcomes of patients with stage I/II breast cancer.

Methods

Patients

We conducted a retrospective study of 63 consecutive patients (aged 42–80 years) with a confirmed histological diagnosis of breast cancer who underwent initial surgery at the Hospital Italiano of Buenos Aires, Argentina. Patients were included if they were diagnosed with stage I/II invasive ductal breast cancer according to the International Union Against Cancer TNM classification system [16] and ≥10 years after surgery. Exclusion criteria included neoadjuvant therapies, lack of tissue, and another primary tumor. After surgery, all patients were treated with the indicated therapy, depending on their clinical status and the histopathological characteristics of their tumor, which were determined according to the recommendations of the European Society for Medical Oncology [17]. The Instituto de Biología y Medicina Experimental and the Hospital Italiano Ethics Committees approved this study, and informed consent was obtained from patients or the relatives of deceased patients, in accordance with the principles of the Helsinki Declaration. Physicians who were unaware of the pathology results acquired clinical information from patients’ medical records, and the anonymity of the data was ensured using a code made available only to the biostatistician.

Tumor samples

Breast tissues embedded in paraffin blocks and fixed in 10% neutral-buffered formalin were retrieved from the surgical archives, and 4-μm thick sections were used in the experiments described below.

Analysis of protein expression

These tissues were processed and immunohistochemistry was used to determine the levels of ligands and receptors in TEpC and in spindle-shaped stromal cells, not associated with the vasculature, and it was completed as described in a previously work [15].

Immunoreactivity was reviewed and scored independently by two pathologists who were blinded to patient outcomes. In uncertain cases, re-evaluation was performed using a double-headed microscope, and staining was evaluated until a consensus was achieved. The agreement in immunohistochemical evaluation between the two observers was 91.77% (Cohen’s kappa coefficient = 0.895). Each sample was assayed in duplicate and was initially examined at 100× magnification followed by observation of five representative fields at 400× magnification along a projected Z-line. Expression levels were evaluated separately for the TEpCs and spindle-shaped stromal cells, not associated to the vasculature, per the percentage of positive cells and staining intensity, which were estimated according to the Allred score [15, 16]. The percentages of positive cells were assigned scores as follows: 0 (<10%), 1 (10%–30%), 2 (31%–60%), 3 (61%–90%), and 4 (>90%). Staining intensity was scored as 0 (no staining), 1 (weak), 2 (moderate), and 3 (strong), according to the relative intensity of staining of TEpCs analyzed using the anti-cytokeratin antibody [15, 18]. The final staining score was calculated using the sum of the percentage of positive cells and the staining intensity score, which ranged from 0 to 7. Stromal cells included in this study had a spindle shape and were not associated with vasculature. CD34 expression was undetectable in this type of stromal cells as previously reported [18].

Patients’ clinicopathological characteristics

Classical prognostic markers were categorized according to cut-offs used in the protocols of the Hospital Italiano, [17] including: a) age < 50 or ≥50 years; b) tumor size <2 or ≥2 cm; c) histological grade according to the Scarff-Bloom-Richardson grading system [19], which is expressed as differentiated (G1), intermediate (G2), and poor (G3); d) expression of estrogen/progesterone receptors and HER2/neu was defined as negative or positive according to Wernicke et al. [17]; and e) presence of regional metastatic lymph nodes was recorded as negative (negative nodes in axillary dissection or sentinel lymph node) or positive (including micrometastasis) (Table 1).

DFS and MFS were defined as the interval from date of surgery to the first observation of tumor occurrence (metastatic occurrence and/or local relapse) and metastatic occurrence, respectively, or last follow-up. The interval from the date of surgery until death or last follow-up was defined as OS. Univariate analyses of DFS, MFS, and OS were performed using the Kaplan-Meier method, and the differences were evaluated using the log-rank (Mantel-Cox) test. When significant variables were identified, we applied the Cox proportional hazards model to the multivariate survival analysis using backward stepwise selection (likelihood ratio) that incorporated only the significant variables. Statistical analysis was performed using SSPS software (version 18.00, Chicago, Illinois) and InfoStat (version 2012, InfoStat Group, National University of Cordoba, Argentina). A two-sided p value <0.05 was considered statistically significant.

Results

The expression of TRAIL was significantly associated with lymph node status (Table 2). High TRAIL expression was detected in 10/40 breast cancer patients with negative lymph nodes, and TRAIL expression was undetectable in 0/16 of patients with positive lymph nodes. SDF-1 expression was significantly associated with tumor size and was high in 31/44 patients with tumors <2 cm and in 7/17 patients with tumors ≥2 cm (p = 0.004, Table 2). High levels of CCL-2 expression were detected in some patients with negative (3/15) or positive (1/42) ER expression (Table 2). The DFS of patients with high CCL-2 expression was 67.7 ± 32.0 months, compared with 123.15 ± 8.28 months for those with low/negative CCL-2 expression (p = 0.048, Table 3).

Table 2

Association of the levels of expression of OPG, TRAIL, RANKL, SDF-1, IL-6, and CCL-2 in TEpCs with the clinicopathological characteristics of patients with early invasive

Characteristics

Ligands in TEpC

OPG

TRAIL

RANKL

SDF-1

IL-6

CCL-2

High expression %

p

High expression %

p

High expression %

p

High expression %

p

High expression %

p

High expression %

p

Age (years)

<50

10.0

>0.999

2.2

0.657

70.0

>0.999

40.0

0.153

22.2

0.464

22.2

0.110

≥50

15.7

16.0

73.1

67.3

39.2

4.1

Tumor size (cm)

<2

13.9

>0.999

17.1

>0.999

65.9

0.114

70.4

0.044*

42.9

0.236

7.3

>0.999

≥2

11.8

17.6

88.2

41.2

23.5

6.2

Histological grade

G1

20.0

0.713

21.4

0.744

46.7

0.073

80.0

0.093

42.9

0.307

6.7

0.357

G2

10.0

20.0

80.9

66.7

45.0

0.0

G3

12.5

13.0

79.2

45.8

25.0

13.0

HER2/neu status

Negative

8.1

0.239

18.9

0.733

76.3

0.388

63.2

>0.999

39.5

0.780

2.9

0.287

Positive

21.7

14.3

65.2

60.9

33.33

13.6

ER status

Negative

13.3

>0.999

7.7

0.429

80.0

0.523

40.0

0.064

42.9

0.753

20.0

0.049*

Positive

13.3

20.0

69.6

69.6

35.6

2.4

PR status

Negative

14.3

>0.999

8.3

0.444

78.6

0.737

50.0

0.351

38.5

>0.999

7.1

>0.999

Positive

13.0

19.6

70.2

66.0

37.0

7.0

Regional lymph nodes

Negative

11.6

0.666

25.0

0.048*

67.4

0.352

58.1

0.555

39.0

0.761

4.7

0.349

Positive

20.0

0.0

81.2

68.7

31.2

14.3

Metastatic occurrence

Negative

20.9

0.180

21.4

0.664

70.4

0.707

59.1

>0.999

30.2

>0.999

2.4

0.109

Positive

18.2

10.0

81.8

63.6

40.0

18.2

Local relapse

Negative

6.1

0.330

20.8

0.576

70.0

0.307

60.0

>0.999

38.8

0.284

4.3

0.266

Positive

20.0

0.0

100.0

60.0

0.0

20.0

Table 3

Univariate analysis of disease-free, metastasis-free, and overall survival of patients with early invasive ductal breast cancer

Univariate

p-value

Disease-free survival

Metastasis-free survival

Overall survival

Age

0.598

0.448

0.500

Tumor size

0.113

0.020*

0.069

Histological grade

0.178

0.291

0.207

HER2/neu status

0.536

0.293

0.103

ER status

0.336

0.191

0.175

PR status

0.691

0.946

0.521

Regional lymph nodes

0.595

0.805

0.620

OPG/TEpC

0.167

0.052

0.178

TRAIL/TEpC

0.465

0.648

0.304

RANKL/TEpC

0.156

0.267

0.307

SDF-1/TEpC

0.660

0.932

0.710

IL-6/TEpC CCL-2

0.873

0.710

0.487

CCL-2/TEpC

0.048*

0.071

0.507

TRAIL-R1/TEpC

0.536

0.339

0.626

TRAIL-R2/TEpC

0.894

0.749

0.392

TRAIL-R3/TEpC

0.009*

0.012*

0.015*

TRAIL-R4/TEpC

0.186

0.131

0.478

RANK/TEpC

0.546

0.991

0.804

CXCR-4/TEpC

0.164

0.255

0.175

IL-6R/TEpC

0.391

0.540

0.626

CCR-2/TEpC

0.013*

0.002*

0.049*

OPG/stromal cells

0.318

0.101

0.441

TRAIL/stromal cells

0.284

0.084

0.337

RANKL/stromal cells

0.139

0.052

0.222

SDF-1/stromal cells

0.792

0.734

0.306

IL-6/stromal cells CCL-2

0.218

0.093

0.168

CCL-2/stromal cells

0.104

0.076

0.505

Association of expression in TEpCs of TRAIL-R1–4, RANK, CXCR-4, IL-6R, and CCR-2 with patients’ clinicopathological characteristics

IL-6R expression in TEpCs was associated with age (Table 4). Specifically, IL-6R expression was higher in 6/8 patients <50 years of age and in 15/48 patients ≥50 years of age (Table 4). Patients with high expression of TRAIL-R3 and CCR-2 in TEpCs were at significantly higher risk for metastatic tumors than patients with low expression (Table 4). High levels of TRAIL-R3 were expressed in 7/11 breast cancer patients with metastasis and in 12/45 patients with non-metastatic tumors (p = 0.032, Table 4). Certain patients with metastatic (5/11) or non-metastatic tumors (2/42) expressed high levels of CCR-2 (Fig. 1 and Table 4). There was an association of high TRAIL-R3 expression with shorter DFS, MFS, and OS (Table 3). The values of DFS, MFS and OS of patients with high TRAIL-R3 expression were as follows (months): 90.04 ± 14.64, 97.02 ± 14.08 and 112.75 ± 12.73, respectively; for patients with low/negative expression were 136.22 ± 7.52, 140.22 ± 6.61 and 146.51 ± 5.16, respectively (Fig. 2 and Table 3).

Table 4

Association of the levels of expression of TRAIL-R1-R4, RANK, CXCR-4, IL-6R, and CCR-2 in TEpCs with the clinicopathological characteristics of patients with early invasive ductal breast cancer

Characteristics

Receptors in TEpC

TRAIL-R1

TRAIL-R2

TRAIL-R3

TRAIL-R4

RANK

CXCR-4

IL-6-R

CCR-2

High expression %

p

High expression %

p

High expression %

p

High expression %

p

High expression %

p

High expression %

p

High expression %

p

High expression %

p

Age (years)

<50

87.5

0.249

0.0

>0.999

40.0

0.729

80.0

0.175

30.0

0.176

50.0

0.259

75.0

0.042*

11.1

>0.999

≥50

63.5

9.6

34.0

54.7

55.8

73.1

31.3

12.0

Tumor size (cm)

<2

66.7

>0.999

9.3

>0.999

35.6

>0.999

57.8

>0.999

50.0

0.579

68.2

>0.999

31.6

0.365

11.9

>0.999

≥2

64.7

5.9

35.3

58.8

58.8

70.6

47.1

12.5

Histological grade

G1

71.4

0.397

13.3

0.245

33.3

>0.999

60.0

0.803

53.3

>0.999

66.7

0.941

38.5

0.078

20.0

0.597

G2

55.0

0.0

36.7

63.6

52.4

71.4

16.7

10.0

G3

75.0

12.5

37.5

54.2

50.0

66.7

50.0

8.7

HER2/neu status

Negative

59.5

0.255

5.4

0.361

25.6

0.054

51.3

0.190

52.6

>0.999

71.0

0.776

26.5

0.083

16.7

0.235

Positive

77.3

13.0

52.2

69.6

52.2

65.2

52.4

4.5

ER status

Negative

66.7

>0.999

13.3

0.590

53.3

0.125

60.0

>0.999

60.0

0.562

73.3

0.757

46.7

0.361

20.0

0.359

Positive

65.9

6.7

29.8

57.4

50.0

67.4

32.5

9.3

PR status

Negative

64.3

>0.999

14.3

0.581

42.9

0.539

57.1

>0.999

57.1

0.766

78.6

0.516

35.7

>0.999

14.3

>0.999

Positive

66.7

6.5

33.3

58.3

51.1

66.0

36.6

11.4

Regional lymph nodes

Negative

64.3

0.342

9.3

>0.999

36.4

>0.999

61.4

0.771

55.8

0.558

72.1

0.533

35.0

0.749

11.9

>0.999

Positive

80.0

6.7

37.5

56.2

43.7

62.5

42.9

13.3

Metastatic occurrence

Negative

66.7

0.108

4.6

0.502

26.7

0.032*

51.1

0.092

50.0

>0.999

70.4

0.473

30.8

0.171

4.8

0.003*

Positive

90.9

9.1

63.6

81.8

45.4

54.5

54.5

45.4

Local relapse

Negative

70.8

>0.999

4.1

0.257

31.4

0.323

56.9

>0.999

48.0

0.669

70.0

0.316

35.6

>0.999

14.6

0.601

Positive

80.0

20.0

60.0

60.0

60.0

40.0

40.0

0.0

Fig. 1

Heat map of the association of ligand and receptor expression in TEpCs and spindle-shaped stromal cells with metastasis. Graphic show data for tumor samples with high and negative/low expression of ligand and receptor

Fig. 2

Association of TRAIL-R3 expression in TEpCs with DFS, MFS, and OS. Kaplan–Meier curves show representative data for tumor samples with high and negative/low expression of TRAIL-R3 in TEpCs. Original magnification: 400×. Scale bars =50 μm

Furthermore, there was an association of high CCR-2 expression with shorter DFS, MFS and OS (Table 3). The values of DFS, MFS, and OS of patients with high CCR-2 expression were as follows (months): 87.57 ± 18.57, 87.71 ± 18.58, and 114.67 ± 15.29, respectively; for patients with low/negative expression were 127.57 ± 8.42, 133.94 ± 7.52, and 140.44 ± 6.41, respectively (Fig. 3 and Table 3).

Fig. 3

Association of CCR-2 expression in TEpCs with DFS, MFS, and OS. Images show representative data of tumor samples with high and negative/low expression of CCR-2 in TEpCs. Original magnification: 400×. Scale bars =50 μm

SDF-1 expression in spindle-shaped stromal cells was associated with histological grades, and high SDF-1 expression was detected in 10/15, 14/21, and 8/24 patients with differentiation grades G1, G2, and G3, respectively (Table 5). In contrast, high expression of OPG and CCL-2 in stromal cells was associated with a higher risk of metastasis (Fig. 1 and Table 5). High expression of OPG was observed in 7/10 patients with metastatic tumors and in 14/43 patients with non-metastatic tumors (p = 0.038, Fig. 1 and Table 5). In patients with metastatic or non-metastatic tumors, 4/11 and 4/44 expressed high levels of CCL-2, respectively (Fig. 1 and Table 5).

Table 5

Association of the levels of expression of OPG, TRAIL, RANKL, SDF-1, IL-6, and CCL-2 in spindle-shaped stroma cells (not associated with the vasculature) with the clinicopathological characteristics of patients with early invasive ductal breast cancer

Characteristics

Ligands in spindle-shaped stromal cells

OPG

TRAIL

RANKL

SDF-1

IL-6

CCL-2

High expression %

p

High expression %

p

High expression %

p

High expression %

p

High expression %

p

High expression %

p

Age (years)

<50

50.0

0.742

66.7

0.137

70.0

0.709

40.0

0.493

22.2

0.664

22.2

0.646

≥50

44.0

35.3

75.0

55.8

17.6

16.3

Tumor size (cm)

<2

48.8

0.255

38.1

>0.999

68.2

0.193

56.8

0.391

21.4

0.483

19.5

0.708

≥2

31.2

41.2

88.2

41.2

11.8

12.5

Histological grade

G1

60.0

0.336

35.7

0.518

60.0

0.491

66.7

0.042*

21.4

0.741

33.3

0.116

G2

35.0

50.0

76.2

66.7

20.0

5.3

G3

39.1

33.3

79.2

33.3

12.5

17.4

HER2/neu status

Negative

44.4

>0.999

44.7

0.272

78.9

0.368

57.9

0.302

18.4

>0.999

17.1

>0.999

Positive

43.5

28.6

65.2

43.5

19.0

18.2

ER status

Negative

46.7

>0.999

42.9

0.761

80.0

0.738

53.3

>0.999

21.4

>0.999

20.0

>0.999

Positive

43.2

37.8

71.7

52.2

17.8

16.7

PR status

Negative

42.9

>0.999

30.8

0.540

78.6

0.742

50.0

>0.999

15.4

>0.999

7.1

0.422

Positive

44.4

41.3

72.3

53.2

19.6

20.9

Regional lymph nodes

Negative

46.1

0.547

41.5

>0.999

74.4

0.745

55.8

0.773

17.1

>0.999

19.0

0.724

Positive

35.7

37.5

68.7

50.0

18.7

14.3

Metastatic occurrence

Negative

32.5

0.038*

34.9

0.072

65.9

0.146

47.3

0.745

11.6

0.163

9.1

0.049*

Positive

70.0

70.0

90.9

54.5

30.0

36.4

Local relapse

Negative

39.6

>0.999

42.9

0.633

72.0

0.621

50.0

>0.999

16.3

0.612

14.9

>0.999

Positive

40.0

25.0

60.0

40.0

0.0

20.0

Univariate analysis of the association of classical prognostic markers with DFS, MFS, and OS

Multivariate analysis

Multivariate analysis of DFS, MFS, and OS of patients with early invasive ductal breast cancer

Variables

HR

95% CI

p

Disease-free survival

TRAIL-R3 in TEpC

3.566

1.164–10.920

0.026

Metastasis-free survival

Tumor size

8.210

2.013–33.477

0.003

CCR-2 in TEpC

10.257

2.569–40.947

0.001

Overall survival

TRAIL-R3 in TEpC

5.741

1.113–29.621

0.037

C.I. confidence interval, HR hazard ratio

Discussion

Tumor progression is a multistep process involving interactions between tumor cells and spindle-shaped stromal cells, not associated with the vasculature, which supply signals that may promote tumor progression [15].

Here we show that high TRAIL expression in TEpCs was significantly associated with negative lymph-node status. Paracrine signaling induced by the binding of TRAIL to the death receptors TRAIL-R1 and TRAIL-R2 induces apoptosis [20–22]. Thus, the association of TRAIL expression in TEpCs of patients with negative lymph nodes might reflect the apoptotic effects of TRAIL that delay tumor progression as well as the extravasation of tumor cells to regional lymph nodes [23].

Patients with TEpCs that expressed high levels of TRAIL-R3 harbored metastases and experienced shorter DFS, MFS, and OS. TRAIL-R3 competes with TRAIL-R1, TRAIL-R2, or both for the binding of TRAIL, which inhibits apoptotic signaling [20]. Moreover, the expression of TRAIL-R3 in TEpCs was an independent prognostic marker for DFS and OS. These findings indicate the importance of evaluating TRAIL-R3 expression in TEpCs, because TRAIL is used to treat tumors. Thus, outcomes may be adversely affected by the level of TRAIL-R3 activity in tumors as well as in the tumor microenvironment.

In contrast, we found that high SDF-1 expression in TEpCs was significantly associated with tumor size <2 cm, which is consistent with the findings of previous studies [24, 25]. Furthermore, high expression of SDF-1 in spindle-shaped stromal cells, not associated with the vasculature, was significantly associated with conventional prognostic markers of less adverse tumor phenotypes, such as low histological grade (G1 and G2).

We show here that the expression of CCL-2 in TEpCs was associated with negative ER-status, which agrees with reports demonstrating that CCL-2 is overexpressed in ER-negative compared with ER-positive tumors [26]. These data suggest the involvement of CCL-2 in the progression of ER-negative breast tumors. Moreover high CCL-2 expression in TEpCs was significantly associated with DFS. CCL-2 directly promotes the malignant phenotype (epithelial mesenchymal transition) of TEpCs and increases their ability to migrate, proliferate, and invade tissues [27–30]. Also, patients with high CCR-2 expression in TEpCs experienced shorter DFS, MFS, and OS. Furthermore, the expression of CCR-2 is up-regulated in breast tumor cells, and knockdown of CCR-2 expression inhibits breast tumor development [31]. Additionally, we show here that CCR-2 expression was an independent prognostic factor for MFS.

Stromal cells such as fibroblast that produce CCL-2 enhance the invasiveness and metastatic growth of human breast cancer cell lines [31], which is consistent with the present findings of a significant association between high CCL-2 expressions in spindle-shaped stromal cells in patients with metastatic early-stage breast cancer. Our data indicate the importance of evaluating CCR-2 expression in TEpCs as well as CCL-2 expression in TEpC and spindle-shaped stromal cells, because the pathways that produce CCR-2 and its ligands may provide targets for the prevention of breast cancer progression and metastasis [29]. Interestingly, we found previously that the expression of CCL-2 in spindle-shaped stromal cells, not associated with the vasculature, correlated positively with the expression of CCR-2 in TEpCs, suggesting that CCL-2 signaling through CCR-2 may contribute to the interactions between TEpCs and spindle-shaped stromal cells, which enhance the malignant phenotype of tumor cells during the early stages of disease [15].

We uncovered a significant association between high OPG expression in spindle-shaped stromal cells and the presence of metastatic breast tumors. This finding is consistent with those showing that OPG produced by a breast tumor induces angiogenesis and inhibits TRAIL-mediated apoptosis to promote the growth of the primary tumor as well as metastatic cells [32, 33].

To our knowledge, this study is the first to demonstrate that high expression of TRAIL-R3 and CCR-2 in TEpCs serves as a prognostic marker of metastatic tumors as well as DFS, MFS, and OS in women with stage I/II invasive breast cancer. These new findings provide a rationale for further studies designed to target TRAIL-R3 and CCR-2 signaling pathways to facilitate the diagnosis, prevention, and treatment of breast cancer.

Conclusions

High levels of TRAIL-R3 and CCR-2 expression in TEpCs identified early breast cancer patients with poor outcomes, including a higher risk of metastasis and shorter DFS, MFS, and OS and represent new independent prognostic factors that may also be suitable therapeutic targets.

Availability of data and materials

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

Authors’ contrib

VL contributed to the conception and design of the study, performed the immunoassays and statistical analysis, and helped to draft the manuscript. LMM participated in the design of the study, performed the statistical analysis, coordination and helped to draft the manuscript. MLC performed the statistical analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis). KMD acquired clinical data and evaluation of the samples. HGR acquired clinical data and evaluation of the samples. AW acquired clinical data and evaluation of the samples. LF coordinated the study and helped to draft the manuscript. AM carried out the immunoassays. MBG contributed to the statistical analysis. FRB helped to draft the manuscript and analyze the data. HC helped to draft the manuscript and analyze the data. HSC helped to draft the manuscript and analyze the data. NAC participated in the design, conception, and conduct of the study and helped to draft the manuscript. All authors read and approved the final version of the manuscript.

Competing interests

The authors declare that they have no conflict of interest.

Consent for publication

Written informed consents were obtained from the patient for publication of this case series. A copy of the written consent is available for review by the Editor of this journal.

Ethics approval and consent to participate

This study was approved by the ethics committee of Hospital Italiano and Instituto de Biología y Medicina Experimental (IBYME) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and conducted according to the Declaration of Helsinki.

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